PCR-Based Pooling of Dried Blood Spots for Detection of Malaria Parasites: Optimization and Application to a Cohort of Ugandan Children

Abstract

Sensitive, high-throughput methods to detect malaria parasites in low-transmission settings are needed. PCR-based pooling strategies may offer a solution. We first used laboratory-prepared samples to compare 2 DNA extraction and 4 PCR detection methods across a range of pool sizes and parasite densities. Pooled Chelex extraction of DNA, followed by nested PCR of cytochrome b, was the optimal strategy, allowing reliable detection of a single low-parasitemic sample (100 parasites/l) in pool sizes up to 50. This PCR-based pooling strategy was then compared with microscopy using 891 dried blood spots from a cohort of 77 Ugandan children followed for 2 years in an urban setting of low endemicity. Among 419 febrile episodes, 35 cases of malaria were detected using the PCR-based pooling strategy and 40 cases using microscopy. All five cases of malaria not detected by PCR were from samples stored for >2 years with parasitemia of <6,000/l, highlighting the issue of possible DNA degradation with long-term storage of samples. Among 472 samples collected from asymptomatic children as part of routine surveillance, 15 (3.2%) were positive by PCR-based pooling compared to 4 (0.8%) by microscopy (P ‫؍‬ 0.01). Thus, this PCR-based pooling strategy for detection of malaria parasites using dried blood spots offers a sensitive and efficient approach for malaria surveillance in low-transmission settings, enabling improved detection of asymptomatic submicroscopic infections and dramatic savings in labor and costs.Due to large-scale implementation of effective control measures, many countries where malaria is endemic are experiencing dramatic declines in disease burden. With this success has come a shift in the end goal from control to elimination (9, 10). However, when the goal is elimination, accurate detection of persons infected with malaria parasites becomes essential (11). Standard surveillance systems depend on diagnosis by microscopy, a method that is technically challenging, labor-intensive, and often inaccurate in operational settings. More recently available rapid diagnostic tests (RDTs) provide convenience and ease of use, but they have limitations in specificity, sensitivity, species identification, and cost (22).PCR-based methods for malaria parasite detection are relatively simple and provide improved sensitivity compared to microscopy and RDTs, especially in settings where infections have low parasitemia or contain mixed species (22). PCR can also be performed on dried blood spots, which are convenient for collection, storage, and transport. Nonetheless, PCR has a long turnaround time, making it an impractical tool for clinical care of individual patients. One exception would be real-time PCR, which has a short turnaround time but carries with it cost and capacity constraints that make it unavailable for rapid diagnosis in most settings. However, there is a potential role for PCR in situations where large numbers of samples are being screened: a high-throughput system could allow accurate, rapid, and cost-effective ass...

Evaluation of Two Nested PCR-Based Diagnostic Assays for Plasmodium Falciparum Infection

“…The result of this study indicated that the nested PCR protocol A was more efficacy and superior for detection of P. falciparum over protocol B. The detection limit of this protocol was good and similar as found in previous nested PCR studies [5,[23][24][25].…”

The Effect of Storage and Extraction Methods on Amplification of Plasmodium falciparum DNA from Dried Blood Spots

“…10 A likely explanation is that some DNA is lost during binding and elution in the spin column. A caveat to this finding is that we only evaluated three PCR assays; assays more sensitive to inhibitors may benefit from use of highly purified DNA obtained from the spin-column technique.…”

“…Extracted DNA was stored at −20 C until use. Nested PCR for cytochrome B mitochondrial DNA using 5 μL template DNA followed previously published methods, 10 except that for round one of amplification primers were CB1ab (5 -TTTAGCAAGTCGATATACACCAGA-3 ) and CB2ab (5 -CTTTAACTTGCCAACTCCCTATCA-3 ); and temperature cycling conditions were: 5 minutes at 94 C, 40 cycles at 94 C for 30 seconds, 62.5 C for 90 seconds, 68 C for 90 seconds, and a final elongation at 68 C for 10 minutes, yielding an amplicon length of 1241 base pairs. Single round PCR of microsatellites was performed for two previously published loci (TA40 and PfG377) using 1 μL of template DNA in a 5 μL reaction volume.…”

Active Case Detection with Pooled Real-Time PCR to Eliminate Malaria in Trat Province, Thailand

“…Pooling samples before analysis facilitates the large-scale application of this technique for surveillance by reducing cost and analysis time. 7,8 As Thailand aims for malaria elimination, including elimination of artemisininresistant parasites, improvements in case detection are necessary. We aimed to determine if pooled real-time PCR could be integrated with the existing active case detection systems in Thailand and if so, if it would be more effective than microscopy for identifying low-density parasitemias.…”